Common ice plant (Mesembryanthemum crystallinum L.) is an annual plant belonging to the genus Mesembryanthemum in the family Aizoaceae and a halophyte capable of growing in seawater-containing soil. Generally, a halophyte refers to a special plant with high salt tolerance which is capable of growing in the soil containing 100 mM NaCl where ordinary plants (glycophyte) can not grow. Common ice plant has a high capacity of absorbing metals such as Cd and Cu, and inorganic salts such as NaCl. For example, it is said that common ice plant is capable of absorbing about 15 g of NaCl per plant. This corresponds to 200 g/m2 or 2 t/ha.
At present, about 10% of the total agricultural area in the world suffers from salt accumulation which makes agriculture difficult. In Japan, there is the same problem. In particular, in Saga Prefecture the problem is more serious because as large as about 65% of its agricultural area is originally reclaimed land.
Common ice plant switches its mode of photosynthesis from C3 (which is seen in soybean, rice, etc.) to CAM (which is seen in orchid and cactus, etc.) under unfavorable environments such as salts, high light and drought. The common ice plant has become a target of various studies as a model plant for elucidating the mechanisms of changes in photosynthesis and the abiotic stress tolerance. Recently, as performed in well-known model plants such as rice and Arabidopsis thaliana, analysis of the whole nucleotide sequence of the genomic DNA is going to be performed in the common ice plant. Therefore, in the near future when functional analysis of various genes or creation of transgenic ice plants will be performed based on the determined nucleotide sequence, transformation of common ice plant will be essential as in the cases of other model plants. Further, for such purposes, an efficient transformation protocol of the common ice plant will be extremely important.
As methods for transforming plants, generally, direct methods such as particle bombardment, electroporation and the polyethylene glycol (PEG) method, and indirect methods such as a gene transfer using a microorganism belonging to the genus Agrobacterium (hereinafter, sometimes simply referred to as “an Agrobacterium”) are known. However, particle bombardment has the following problems: (i) gene transfer is limited to surface cells; (ii) recombination and deletion are apt. to occur in the final transfered fragments; (iii) transformed plants often turn out chimeric; (v) expensive instruments are necessary; (v) since fine metal particles are scattered, human body may be endangered. Electroporation has the following problems: (i) since DNA transfer through cell walls is not easy as in bacteria, decrease in transfer efficiency and cell damage are unavoidable; (ii) since protoplast is the target for gene transfer, electroporation is inapplicable to those plant species in which a protoplast-to-plantlet regeneration system has not been established; thus, application frequency is extremely low; (iii) since a long culture period is required, mutation ratio increases and the probability to obtain normal transformants of interest decreases. The PEG method has similar problems to those seen in electroporation (e.g., decrease in transfer efficiency (actually, the efficiency further decreases) and cell damage are unavoidable). Therefore, at present, it is believed that gene transfer using an Agrobacterium is most secure and useful. In this method, it is possible to integrate a DNA of interest (recombinant gene, etc.) into the plant genome by using the Agrobacterium itself as a biological vector and utilizing the recombinant sequence (T-DNA region) and recombinant enzymes possessed by this bacterium.
Gene transfer using an Agrobacterium has been established to date in various plant species, and a large number of stable transformants have been produced.
With respect to the common ice plant, however, gene transfer into root apex cells and cultured cells (Andolfatto et al., Physiol. Plant., vol. 90, pp. 708-714 (1994); Ishimaru, Plant Cell Tissue Organ Culture, vol. 57, pp. 61-63 (1999)) suggest that Agrobacterium is able to infect the common ice plant cells (i.e., transfer of a foreign gene is possible), subsequent regeneration ratios were extremely low (Cushman et al., Plant Cell Rep., vol. 19, pp. 459-463 (2000)) and stable transformants have not yet been achieved.